RESUMO
Bacterial homologous lysine and arginine decarboxylases play major roles in the acid stress response, physiology, antibiotic resistance and virulence. The Escherichia coli enzymes are considered as their archetypes. Whereas acid stress triggers polymerisation of the E. coli lysine decarboxylase LdcI, such behaviour has not been observed for the arginine decarboxylase Adc. Here we show that the Adc from a multidrug-resistant human pathogen Providencia stuartii massively polymerises into filaments whose cryo-EM structure reveals pronounced differences between Adc and LdcI assembly mechanisms. While the structural determinants of Adc polymerisation are conserved only in certain Providencia and Burkholderia species, acid stress-induced polymerisation of LdcI appears general for enterobacteria. Analysis of the expression, activity and oligomerisation of the P. stuartii Adc further highlights the distinct properties of this unusual protein and lays a platform for future investigation of the role of supramolecular assembly in the superfamily or arginine and lysine decarboxylases.
Assuntos
Carboxiliases , Providencia , Carboxiliases/genética , Carboxiliases/metabolismo , Escherichia coli/metabolismo , Providencia/enzimologiaRESUMO
The only enzyme responsible for cadaverine production in the major multidrug-resistant human pathogen Pseudomonas aeruginosa is the lysine decarboxylase LdcA. This enzyme modulates the general polyamine homeostasis, promotes growth, and reduces bacterial persistence during carbenicillin treatment. Here we present a 3.7-Å resolution cryoelectron microscopy structure of LdcA. We introduce an original approach correlating phylogenetic signal with structural information and reveal possible recombination among LdcA and arginine decarboxylase subfamilies within structural domain boundaries. We show that LdcA is involved in full virulence in an insect pathogenesis model. Furthermore, unlike its enterobacterial counterparts, LdcA is regulated neither by the stringent response alarmone ppGpp nor by the AAA+ ATPase RavA. Instead, the P. aeruginosa ravA gene seems to play a defensive role. Altogether, our study identifies LdcA as an important player in P. aeruginosa physiology and virulence and as a potential drug target.
Assuntos
Proteínas de Bactérias/química , Carboxiliases/química , Evolução Molecular , Pseudomonas aeruginosa/enzimologia , Fatores de Virulência/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Carboxiliases/genética , Carboxiliases/metabolismo , Microscopia Crioeletrônica , Expressão Gênica , Humanos , Cinética , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Terciária de Proteína , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Polyamines are small amino-acid derived polycations capable of binding negatively charged macromolecules. Bacterial polyamines are structurally and functionally diverse, and are mainly produced biosynthetically by pyridoxal-5-phosphate-dependent amino acid decarboxylases referred to as Lysine-Arginine-Ornithine decarboxylases (LAOdcs). In a phylogenetically limited group of bacteria, LAOdcs are also induced in response to acid stress. Here, we performed an exhaustive phylogenetic analysis of the AAT-fold LAOdcs which showcased the ancient nature of their short forms in Cyanobacteria and Firmicutes, and emergence of distinct subfamilies of long LAOdcs in Proteobacteria. We identified a novel subfamily of lysine decarboxylases, LdcA, ancestral in Betaproteobacteria and Pseudomonadaceae. We analyzed the expression of LdcA from Pseudomonas aeruginosa, and uncovered its role, intimately linked to cadaverine (Cad) production, in promoting growth and reducing persistence of this multidrug resistant human pathogen during carbenicillin treatment. Finally, we documented a certain redundancy in the function of the three main polyamines-Cad, putrescine (Put), and spermidine (Spd)-in P. aeruginosa by demonstrating the link between their intracellular level, as well as the capacity of Put and Spd to complement the growth phenotype of the ldcA mutant.
Assuntos
Carboxiliases/genética , Pseudomonas aeruginosa/genética , Cadaverina/metabolismo , Cianobactérias/genética , Firmicutes/genética , Família Multigênica , Filogenia , Proteobactérias/genética , Pseudomonas aeruginosa/enzimologiaRESUMO
The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions.
